Paralytic Shellfish Toxin Profiles Research Articles

Uptake of Paralytic Shellfish Toxins by Blacklip Abalone (Haliotis rubra rubra Leach) from direct exposure to Alexandrium catenella microalgal cells and toxic aquaculture feed

The Tasmanian abalone fishery represents the largest wild abalone resource in the world, supplying close to 25% of the annual wild-caught global harvest. Prompted by the need to manage Paralytic Shellfish Toxin (PST) contamination of Blacklip Abalone (Haliotis rubra rubra) from east coast Tasmania, the uptake of toxins by this species is investigated in a land-based, controlled aquaculture setting. Abalone were exposed to either live Alexandrium catenella microalgal cultures or PST contaminated feed pellets during a 28 day exposure period and toxins quantified in viscera, foot muscle and epipodium tissues. PST profiles of abalone foot tissues were dominated by saxitoxin and neosaxitoxin, whilst viscera more closely resembled those of the toxin source (A. catenella cells rich in gonyautoxin 1&4 and 2&3 or feed pellets containing A. catenella extracts rich in these analogues). This indicates direct uptake of PST in the viscera via browsing/grazing on the pellet and /or sedimented microalgal cells. After exposure to A. catenella cell culture, PST concentrations in the foot (muscle + epipodium) were on average 8 times higher than in the viscera. Higher toxicity of foot tissue was caused by higher PST content of the epipodium (up to 1,085 µg STX.2HCl equiv. kg−1), which despite its small contribution to total animal weight significantly added to the overall toxin burden. Higher PST levels in the abalone foot suggest that toxin monitoring programmes may not need to routinely analyse both foot and viscera, potentially allowing for a 50% reduction of analytical costs. This option is being further investigated with continuing field studies.

Paralytic Shellfish Toxins (PST)-Transforming Enzymes: A Review.

Paralytic shellfish toxins (PSTs) are a group of toxins that cause paralytic shellfish poisoning through blockage of voltage-gated sodium channels. PSTs are produced by prokaryotic freshwater cyanobacteria and eukaryotic marine dinoflagellates. Proliferation of toxic algae species can lead to harmful algal blooms, during which seafood accumulate high levels of PSTs, posing a health threat to consumers. The existence of PST-transforming enzymes was first remarked due to the divergence of PST profiles and concentrations between contaminated bivalves and toxigenic organisms. Later, several enzymes involved in PST transformation, synthesis and elimination have been identified. The knowledge of PST-transforming enzymes is necessary for understanding the processes of toxin accumulation and depuration in mollusk bivalves. Furthermore, PST-transforming enzymes facilitate the obtainment of pure analogues of toxins as in natural sources they are present in a mixture. Pure compounds are of interest for the development of drug candidates and as analytical reference materials. PST-transforming enzymes can also be employed for the development of analytical tools for toxin detection. This review summarizes the PST-transforming enzymes identified so far in living organisms from bacteria to humans, with special emphasis on bivalves, cyanobacteria and dinoflagellates, and discusses enzymes’ biological functions and potential practical applications.

Detection of Paralytic Shellfish Toxins in Southern Rock Lobster Jasus edwardsii Using the Qualitative Neogen™ Lateral Flow Immunoassay: Single-Laboratory Validation.

Paralytic shellfish toxins (PST) are a significant problem for the Tasmanian shellfish and Southern Rock Lobster (Jasus edwardsii) industries, and the introduction of a rapid screening test in the monitoring program could save time and money. The aim was to perform a single-laboratory validation of the Neogen rapid test for PST in the hepatopancreas of Southern Rock Lobster. The AOAC INTERNATIONAL guidelines for the validation of qualitative binary chemistry methods were followed. Three different PST profiles (mixtures) were used, of which two were commonly found in naturally contaminated lobster hepatopancreas (high in gonyautoxin 2&3 and saxitoxin), and the third toxin profile was observed in a few select animals (high in gonyautoxin 1&4). The Neogen test consistently returned negative results for non-target toxins (selectivity). The probability of detection (POD) of PST in the lobster hepatopancreas using the Neogen test increased with increasing PST concentrations. POD values of 1.0 were obtained at ≥0.57 mg STX-diHCl eq/kg in mixtures 1 and 2, and 0.95 and 1.0 for mixture 3 at 0.79 and 1.21 mg STX-diHCl eq/kg, respectively, with a fitted POD of 0.98 for 0.80 mg STX-diHCl eq/kg. The performance of the Neogen test when using four different production lots (ruggedness) showed no significant differences. The results of the validation study were satisfactory and the Neogen test is being trialed within the Tasmanian PST monitoring program of Southern Rock Lobster. The Neogen rapid kit was successfully validated for the detection of PST in Southern Rock Lobster hepatopancreas.

Paralytic shellfish toxin profiles in mussel, cockle and razor shell under post-bloom natural conditions: Evidence of higher biotransformation in razor shells and cockles

Concentrations of the paralytic shellfish toxins GTX6, C1+2, GTX5, C3+4, dcSTX, dcNEO and dcGTX2+3 were determined by LC-FLD in composite samples of whole soft tissues of mussels (Mytilus galloprovincialis), cockles (Cerastoderma edule) and razor shells (Solen marginatus) after exposure to a Gymnodinium catenatum bloom. Specimens were harvested weekly during three months under natural depuration conditions in the Mira branch of Aveiro lagoon, Portugal. Under the decline of G. catenatum cell densities, elimination or transformation of the uptake toxins associated with the ingestion of toxic cells differed among the surveyed species. Ratio between the toxins dcSTX plus dcGTX2+3 plus dcNEO and toxins GTX6 plus GTX5 plus C1+2 plus C3+4 was used to illustrate the biotransformation occurring in the bivalves. Enhancement of the ratios was observed for razor shells and cockles seven weeks after the peak of the algal bloom. Most likely it reflects more intense biotransformation in razor shells and cockles than in mussels. Conversion into toxins of higher toxicity may prolong the bivalve toxicity. These results show the complexity of toxin elimination in bivalves under post-bloom conditions and emphasize the pertinence of monitoring programs of bivalve toxicity in order to protect human health.

Multi-year assessment of paralytic shellfish toxins in hard clam species along the coastline of Jiangsu Province, China

Paralytic shellfish toxins (PSTs) are notorious neurotoxins that threaten public health and food safety worldwide. Although PST monitoring programs have recently been established throughout China, the profiles and variation of PSTs in important commercial clams (e.g., Mactra veneriformis, Ruditapes philippinarum, and Meretrix meretrix) along the Jiangsu Province coastline remain largely unexplored. In this study, a validated hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) method was used to examine PST profiles and levels in 540 clam samples from natural production areas along Jiangsu Province coastline during 2014–2016. Although the PST levels (≤6.38 μg saxitotoxin equivalents (eq)/kg) were consistently below European Union regulatory limits (≤800 μg saxitotoxin eq/kg) during this time period, saxitotoxin, decarbamoylsaxitotoxin, and gonyautoxins 1 and 4 were detected, and nearly 40% of the samples were saxitotoxin-positive. The PST levels also varied significantly by seasons, with peak values observed in May during 2014–2016. This is the first systematic report of PSTs in clams from Jiangsu Province, and additional research and protective measures are needed to ensure the safety of clams harvested in this area.

Cryptic diversity within the Scytonema complex: Characterization of the paralytic shellfish toxin producer Heterosyctonema crispum, and the establishment of the family Heteroscytonemataceae (Cyanobacteria/Nostocales)

Strains of the freshwater filamentous, benthic cyanobacterium Scytonema crispum Agardh isolated from six sites in subtropical south-east Queensland were characterised using a combination of phenotypic and genetic traits. Morphologically, the strains were consistent with the description of Scytonemataceae sensu stricto, and the description of Scytonema crispum. However, phylogenetic analysis of the 16S rRNA gene, the 16S-23S rRNA operon, and the nifH gene revealed that these strains and three others from outside Australia formed a monophyletic clade distinct from Scytonema and other species in the Scytonemataceae. Collectively, this data suggests this group is sufficiently evolutionarily distinct to be placed in a new family, Heteroscytonemataceae fam. nov. Accordingly, the taxon previously known as S. crispum has been transferred to a new genus Heteroscytonema gen nov., as H. crispum. Some strains of H. crispum exhibited facultative production of paralytic shellfish toxins (PSTs). The concentration of PSTs produced by individual strains varied widely, from 2.7 μg g−1 to 171.3 μg g−1, and included C toxins, decarbamoyl saxitoxin (dcSTX), gonyautoxins (GTX2, GTX3 and GTX5), saxitoxin (STX) and uncharacterised PSTs. The majority of the Australian strains produced dcSTX as the dominant saxitoxin analogue, a significant finding given that dcSTX has approximately half the relative toxicity of STX. The PST profile varied within and between Australian strains of H. crispum and in strains collected from New Zealand and the United States. The sxtA gene, one of the determinants for the production of PSTs, was present in all strains in which PSTs were detected. The discovery of PST-producing H. crispum in the headwaters of a major drinking water reservoir presents a serious risk for potential human and animal exposure to these neurotoxic compounds and further highlights the importance of monitoring benthic cyanobacteria populations for potentially toxigenic species.

Re-evaluation of paralytic shellfish toxin profiles in cyanobacteria using hydrophilic interaction liquid chromatography-tandem mass spectrometry

To date Paralytic shellfish toxin (PST) variants in cyanobacteria have primarily been characterized using high performance liquid chromatography coupled with fluorescence detection. In this study we re-evaluated the PST profiles of five cyanobacterial cultures (Dolichospermum circinale AWQC131C, Aphanizomenon sp. NH-5, Raphidiopsis raciborskii T3, Scytonema cf. crispum CAWBG524 and CAWBG72) and one environmental sample (Microseria wollei) using hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry. A total of 35 different PST variants were detected. D. circinale contained the highest number of variants (23), followed by S. cf. crispum CAWBG72 (21). Many of the variants detected in the cultures/environmental sample had not been reported from these strains previously: D. circinale (14 variants), S. cf. crispum CAWBG72 (16), S. cf. crispum CAWBG524 (9), Aphanizomenon sp. (9), R. raciborskii (7), and M. wollei (7). Of particular interest was the detection of M-toxins (Aphanizomenon sp., R. raciborskii, D. circinale). These have previously only been identified from shellfish where they were thought to be metabolites. Well-characterized PST variant profiles are essential for research investigating the genetic basis of PST production, and given that the toxicity of each variants differs, it will assist in refining risk assessments.

Insertions within the Saxitoxin Biosynthetic Gene Cluster Result in Differential Toxin Profiles.

The neurotoxin saxitoxin and related paralytic shellfish toxins are produced by multiple species of cyanobacteria and dinoflagellates. This study investigates the two saxitoxin-producing strains of Scytonema crispum, CAWBG524 and CAWBG72, isolated in New Zealand. Each strain was previously reported to have a distinct paralytic shellfish toxin profile, a rare observation between strains within the same species. Sequencing of the saxitoxin biosynthetic clusters ( sxt) from S. crispum CAWBG524 and S. crispum CAWBG72 revealed the largest sxt gene clusters described to date. The distinct toxin profiles of each strain were correlated to genetic differences in sxt tailoring enzymes, specifically the open-reading frame disruption of the N-21 sulfotransferase sxtN, adenylylsulfate kinase sxtO, and the C-11 dioxygenase sxtDIOX within S. crispum CAWBG524 via genetic insertions. Heterologous overexpression of SxtN allowed for the proposal of saxitoxin and 3'-phosphoadenosine 5'-phosphosulfate as substrate and cofactor, respectively, using florescence binding assays. Further, catalytic activity of SxtN was confirmed by the in vitro conversion of saxitoxin to the N-21 sulfonated analog gonyautoxin 5, making this the first known report to biochemically confirm the function of a sxt tailoring enzyme. Further, SxtN could not convert neosaxitoxin to its N-21 sulfonated analog gonyautoxin 6, indicating paralytic shellfish toxin biosynthesis most likely occurs along a predefined route. In this study, we identified key steps toward the biosynthetic conversation of saxitoxin to other paralytic shellfish toxins.

A review of the global distribution of Alexandrium minutum (Dinophyceae) and comments on ecology and associated paralytic shellfish toxin profiles, with a focus on Northern Europe.

Alexandrium minutum is a globally distributed harmful algal bloom species with many strains that areknown to produce paralytic shellfish toxins (PSTs) and consequently represent a concern to human andecosystem health. This review highlightsthat A.minutum typically occurs in sheltered locations, with cell growth occurring during periods of stable water conditions. Sediment characteristics are important in the persistence of this species within a location, with fine sediments providing cyst deposits for ongoing inoculation to the water column. Toxic strains of A.minutum do not produce a consistent toxin profile, different populations produce a range of PSTs in differing quantities. Novel cluster analysis of published A.minutum toxin profiles indicates five PST profile clusters globally. Some clusters are grouped geographically (Northern Europe) while others are widely spread. Isolates from Taiwan have arange of toxin profile clusters and this area appears to have the most diverse set of PST producing A.minutum populations. These toxin profiles indicate that within the United Kingdom there are two populations of A.minutum grouping with strains from Northern France and Southern Ireland. There is a degree of interconnectivity in this region due tooceanic circulation and a high level of shipping andrecreational boating. Further research into theinterrelationships between the A.minutum populations in this global region would be of value.

Single-Laboratory Validation of the Neogen Qualitative Lateral Flow Immunoassay for the Detection of Paralytic Shellfish Toxins in Mussels and Oysters.

Detection of paralytic shellfish toxins (PSTs) in bivalve shellfish by analytical methods is complicated and costly, requiring specific expertise and equipment. Following extensive blooms of Alexandrium tamarense Group 1 in Tasmania, Australia, an investigation was made into commercially available screening test kits suitable for use with the toxin profiles found in affected bivalves. The qualitative Neogen rapid test kit, with a modified protocol to convert gonyautoxins GTX1&4 and GTX2&3 into neosaxitoxin and saxitoxin (STX), respectively, with higher cross-reactivities, was the best fit-for-purpose. This validation study of the test kit and the modified protocol was undertaken following AOAC INTERNATIONAL guidelines for the validation of qualitative binary chemistry methods. The validation used four different PST profiles representing natural profiles found in Australia and in Europe: two in a mussel matrix and two in an oyster matrix. The test kit was shown to have appropriate selectivity of the toxin analogs commonly found in bivalve shellfish. The matrix and probability of detection (POD) study showed that the rapid test kit used with the modified protocol was able to consistently detect PST at the bivalve regulatory level of 0.8 mg STX⋅2HCl eq/kg, with a POD estimated via the binomial logistic regression of 1.0 at 0.8 mg STX⋅2HCl eq/kg in all tested profiles in both matrixes. The POD at 0.4 mg STX⋅2HCl eq/kg was 0.75 and 0.46 for the two toxin profiles in an oyster matrix and 0.96 and 1.0 for the two toxin profiles in a mussel matrix. No significant differences in the PODs of the PSTs at the regulatory level were found between production lots of the test kits. The results suggest the method is suitable to undergo a collaborative validation study.

Experimental uptake and depuration of paralytic shellfish toxins in Southern Rock Lobster, Jasus edwardsii

In October 2012, paralytic shellfish toxins (PST) were detected in the hepatopancreas of Southern Rock Lobsters (Jasus edwardsii) collected from the east coast of Tasmania, Australia. This resulted in the first commercial closure in Australia for this species. Questions were raised on how the toxins were transferred to the lobsters, how long the toxins would persist, whether PST-contaminated hepatopancreas posed a risk to human health, and what management strategies could be applied. The aim of this study was to investigate whether PST-contaminated mussels are a potential vector enabling toxin accumulation in J. edwardsii and to collect information on toxin uptake, distribution and depuration rates and toxin profiles under controlled experimental settings. Lobsters were fed mussels naturally contaminated with PST for a period of 28 days in an experimental setting; following this, lobsters were allocated to either fed or starved treatment groups. PST were not detected in the tail tissue of lobsters at any stage of the experiment. Lobster hepatopancreas contained mean levels of 2.4 mg STX.2HCl eq/kg after 28 days of uptake, although substantial variability in total toxicity was observed. The PST profile of the hepatopancreas was similar to that of the contaminated mussels used as feed. Significant differences were noted in the PST depuration rates between fed and starved treatment groups. The daily depuration rate for total PST was estimated to be 0.019 and 0.013 mg STX.2HCl eq/kg for the fed and starved treatment groups respectively using a constant-rate decay model. After 42 days of depuration, total PST (STX equivalents) levels in the hepatopancreas of all lobsters were below 0.8 mg STX.2HCl eq/kg, which represents the regulatory level applied to bivalves. This result indicates that long-term holding to depurate PST may potentially be used as a risk management tool.

Bioactive compounds of marine dinoflagellate isolates from western Greenland and their phylogenetic association within the genus Alexandrium

The diversity and biogeography of populations of the toxigenic marine dinoflagellate genus Alexandrium, a major global cause of paralytic shellfish poisoning (PSP), are represented by only a few studies based upon a low number of cultured isolates and remain poorly described in Arctic and sub-Arctic waters. Multiple clonal isolates (n=22) of the Alexandrium tamarense species complex, and a single isolate of A. tamutum, were collected from the water column while on board an oceanographic expedition to the west coast of Greenland. After culturing of these isolates under controlled conditions, their phylogenetic affinities within the genus Alexandrium were characterized by sequence analysis of nuclear large sub-unit (LSU) rDNA. Based upon morphological and molecular genetic criteria, all isolates of the A. tamarense species complex were consistent with membership in the Group I ribotype (previously known as the North American ribotype). Phenotypic signatures were also analyzed based upon their respective profiles of paralytic shellfish toxins (PST) and allelochemical interactions against a target cryptophyte Rhodomonas, as determined by lytic potency. All isolates conforming to the A. tamarense Group I produced PST, but no toxins were detected in A. tamutum P2E2. Unusually, only carbamoyl toxins were produced among the A. tamarense Group I isolates from Greenland; sulfocarbamoyl derivatives, generally present in A. tamarense population from other locations, including the Arctic, North Pacific and North Atlantic, were absent from all isolates. Allelochemical activity, causing cell lysis of Rhodomonas, but generally being unrelated to cellular PST, was expressed by all A. tamarense isolates and also by A. tamutum, but varied widely in potency. Comparison of the genotypic (rDNA) and phenotypic (PST profile, allelochemical activity) characteristics of Greenland isolates with those of other Arctic populations reveals a complex pattern of intra-specific diversity. Estimation of diversity relationships is problematic because of the distinct patterns of divergence and lack of evidence of linkage among the alternative biomarkers and morphology. Nevertheless, such studies are necessary as the basis for constructing hindcasting scenarios and predicting changes in Alexandrium species distribution in the Arctic from the regional to the global scale.

SxtA and sxtG gene expression and toxin production in the Mediterranean Alexandrium minutum (Dinophyceae).

The dinoflagellate Alexandrium minutum is known for the production of potent neurotoxins affecting the health of human seafood consumers via paralytic shellfish poisoning (PSP). The aim of this study was to investigate the relationship between the toxin content and the expression level of the genes involved in paralytic shellfish toxin (PST) production. The algal cultures were grown both in standard f/2 medium and in phosphorus/nitrogen limitation. In our study, LC-HRMS analyses of PST profile and content in different Mediterranean A. minutum strains confirmed that this species was able to synthesize mainly the saxitoxin analogues Gonyautoxin-1 (GTX1) and Gonyautoxin-4 (GTX4). The average cellular toxin content varied among different strains, and between growth phases, highlighting a decreasing trend from exponential to stationary phase in all culture conditions tested. The absolute quantities of intracellular sxtA1 and sxtG mRNA were not correlated with the amount of intracellular toxins in the analysed A. minutum suggesting that the production of toxins may be regulated by post-transcriptional mechanisms and/or by the concerted actions of alternative genes belonging to the PST biosynthesis gene cluster. Therefore, it is likely that the sxtA1 and sxtG gene expression could not reflect the PST accumulation in the Mediterranean A. minutum populations under the examined standard and nutrient limiting conditions.

Bacterial community affects toxin production by Gymnodinium catenatum.

The paralytic shellfish toxin (PST)-producing dinoflagellate Gymnodinium catenatum grows in association with a complex marine bacterial community that is both essential for growth and can alter culture growth dynamics. Using a bacterial community replacement approach, we examined the intracellular PST content, production rate, and profile of G. catenatum cultures grown with bacterial communities of differing complexity and composition. Clonal offspring were established from surface-sterilized resting cysts (produced by sexual crosses of strain GCDE06 and strain GCLV01) and grown with: 1) complex bacterial communities derived from each of the two parent cultures; 2) simplified bacterial communities composed of the G. catenatum-associated bacteria Marinobacter sp. strain DG879 or Alcanivorax sp. strain DG881; 3) a complex bacterial community associated with an untreated, unsterilized sexual cross of the parents. Toxin content (STX-equivalent per cell) of clonal offspring (134–197 fmol STX cell−1) was similar to the parent cultures (169–206 fmol STX cell−1), however cultures grown with single bacterial types contained less toxin (134–146 fmol STX cell−1) than offspring or parent cultures grown with more complex mixed bacterial communities (152–176 fmol STX cell−1). Specific toxin production rate (fmol STX day−1) was strongly correlated with culture growth rate. Net toxin production rate (fmol STX cell−1 day−1) did not differ among treatments, however, mean net toxin production rate of offspring was 8-fold lower than the parent cultures, suggesting that completion of the sexual lifecycle in laboratory cultures leads to reduced toxin production. The PST profiles of offspring cultures were most similar to parent GCDE06 with the exception of cultures grown with Marinobacter sp. DG879 which produced higher proportions of dcGTX2+3 and GC1+2, and lower proportions of C1+2 and C3+4. Our data demonstrate that the bacterial community can alter intracellular STX production of dinoflagellates. In G. catenatum the mechanism appears likely to be due to bacterial effects on dinoflagellate physiology rather than bacterial biotransformation of PST toxins.

First detection of paralytic shellfish poisoning (PSP) toxins in Icelandic mussels (Mytilus edulis): Links to causative phytoplankton species

Paralytic shellfish poisoning (PSP) toxins were detected in blue mussels (Mytilus edulis) from two harvesting areas, Eyjafjordur on the north coast and Breidafjordur on the west coast of Iceland in 2009. During a bloom of Alexandrium spp. at both locations in June of that year, blue mussels were found to be contaminated with paralytic shellfish toxins (PSTs), leading to extensive closures of these harvesting sites.Phytoplankton data taken during this time showed the presence of large numbers of Alexandrium tamarense, with smaller numbers of Alexandrium ostenfeldii also being detected. Mussel samples were analysed by mouse bioassay (MBA) and liquid chromatography with fluorescence detection (LC–FLD). Toxicity over 10 times the European Union (EU) regulatory limit was observed in samples from Eyjafjordur while levels over 4 times this limit were detected in samples from Breidafjordur. The toxin profile determined by LC–FLD was found to be composed primarily of the carbamate toxins gonyautoxin-2,3 (GTX-2,3). Saxitoxin (STX) was also detected in all samples analysed and was the second most abundant toxin present. Gonyautoxin-1,4 (GTX-1,4) was detected at lower concentrations in half the samples analysed from both locations. Comparison is made between predicted toxin profiles from these algal species and the toxin profiles determined through LC–FLD analysis.These results represent the first identification and PST profile determination in shellfish harvested from Icelandic waters.

Investigation of extraction and analysis techniques for Lyngbya wollei derived Paralytic Shellfish Toxins

Paralytic Shellfish Toxins (PSTs) are highly toxic metabolic by-products of cyanobacteria and dinoflagellates. The filamentous cyanobacterium Lyngbya wollei produces a unique set of PSTs, including L. wollei toxins (LWT) 1–6. The accurate identification and quantification of PSTs from Lyngbya filaments is challenging, but critical for understanding toxin production and associated risk, as well as for providing baseline information regarding the potential for trophic transfer. This study evaluated several approaches for the extraction and analysis of PSTs from field-collected L. wollei dominated algal mats. Extraction of PSTs from lyophilized Lyngbya biomass was assessed utilizing hydrochloric acid and acetic acid at concentrations of 0.001–0.1 M. Toxin profiles were then compared utilizing two analysis techniques: pre-column oxidation (peroxide and periodate) High Performance Liquid Chromatography (HPLC) with Fluorescence (FL) detection and LC coupled with Mass Spectrometry (MS). While both acid approaches efficiently extracted PSTs, hydrochloric acid was found to convert the less toxic LWT into the more toxic decarbamoylgonyautoxins 2&3 (dcGTX2&3) and decarbamoylsaxitoxin (dcSTX). In comparison, extraction with 0.1 M acetic acid preserved the original toxin profile and limited the presence of interfering co-extractants. Although pre-chromatographic oxidation with HPLC/FL was relatively easy to setup and utilize, the method did not resolve the individual constituents of the L. wollei derived PST profile. The LC/MS method allowed characterization of the PSTs derived from L. wollei, but without commercially available LWT 1–6 standards, quantitation was not possible for the LWT. In future work, evaluation of the risk associated with L. wollei derived PSTs will require commercially available standards of LWT 1–6 for accurate determinations of total PST content and potency.

Toxin content differs between life stages of Alexandrium fundyense (Dinophyceae)

Different life stages of two mating-compatible clones of the paralytic shellfish toxin (PST)-producing dinoflagellate Alexandrium fundyense Balech were separated using a combination of techniques; culturing and sampling methods were used to separate vegetative cells and gametes, and sorting flow cytometry was used to separate zygotes. PST profiles were significantly different between life stages; the two gonyautoxins GTX1 and 2 were present in vegetative and senescent cells, but disappeared from gametes and zygotes. Toxin-profile changes were shown to occur very quickly in both strains when pellicle cyst formation was induced by shaking (four minutes) followed by rinsing on a screen. These pellicle cysts produced from exponentially-growing, vegetative cells lost GTX1 and 2 completely. Rapid toxin epimerization of GTX1 to GTX4 and GTX2 to GTX3 is one possible explanation, although the biological advantage of this remains unclear. Another possible explanation is that during the mating phase of a bloom or when cells are disturbed, GTX1 and GTX2 are released into the surrounding water. It may be advantageous for a dinoflagellate bloom to be surrounded by free toxins in the water.

Characterization of paralytic shellfish toxins in seawater and sardines (Sardina pilchardus) during blooms of Gymnodinium catenatum

The re-emergence of Gymnodinum catenatum blooms after a 10 year hiatus of absence initiated the present investigation. This study aims to evaluate the exposure of small pelagic fishes to paralytic shellfish toxins (PST) during blooms of G. catenatum. Sardines (Sardina pilchardus) were selected as a representative fish species. In order to assess toxin availability to fish, both intracellular PSTs (toxin retained within the algal cells) and extracellular PSTs (toxin found in seawater outside algal cells) were quantified, as well as toxin levels within three fish tissue matrices (viscera, muscle and brain). During the study period, the highest cell densities of G. catenatum reached 2.5 × 104 cells l−1 and intracellular PST levels ranged from 3.4 to 398 ng STXeq l−1 as detected via an enzyme linked immunosorbent assay (ELISA). Measurable extracellular PSTs were also detected in seawater (0.2–1.1 μg STXeq l−1) for the first time in Atlantic waters. The PST profile in G. catenatum was determined via high performance liquid chromatography with fluorescence detection (HPLC-FLD) and consisted mostly of sulfocarbamoyl (C1+2, B1) and decarbamoyl (dcSTX, dcGTX2+3, dcNEO) toxins. The observed profile was similar to that reported previously in G. catenatum blooms in this region before the 10-year hiatus. Sardines, planktivorous fish that ingest a large number of phytoplankton cells, were found to contain PSTs in the viscera, reaching a maximum of 531 μg STXeq kg−1. PSTs were not detected in corresponding muscle or brain tissues. The PST profile characterized in sardine samples consisted of the same sulfocarbamoyl and decarbamoyl toxins found in the algal prey with minor differences in relative abundance of each toxin. Overall, the data suggest that significant biotransformation of PSTs does not occur in sardines. Therefore, planktivorous fish may be a good tracer for the occurrence of offshore G. catenatum blooms and the associated PSTs produced by these algae.

Sam, Sahh and Map gene expression during cell division and paralytic shellfish toxin production of Alexandrium catenella (Dinophyceae)

L.D. Harlow, A. Negri, G.M. Hallegraeff and A. Koutoulis. 2007. Sam, Sahh and Map gene expression during cell division and paralytic shellfish toxin production of Alexandrium catenella (Dinophyceae). Phycologia 46: 666–674. DOI: 10.2216/07-25.1The expression of genes potentially involved in paralytic shellfish toxin (PST) biosynthesis was quantified during cell division of the dinoflagellate Alexandrium catenella. Basic precursors of the neurotoxic PST molecule(s), such as S-adenosylmethionine (SAM), have been hypothesised, but no genes coding for PST production have been definitively identified in dinoflagellates. Sam encodes the enzyme SAM synthetase and two other candidate PST genes S-adenosylhomocysteine hydrolase (Sahh) and methionine aminopeptidase (Map) have been previously identified in the closely related PST producing dinoflagellate Alexandrium fundyense. PST production and expression of Sam, Sahh and Map were examined concurrently during cell division in A. catenella. The PST profile of A. catenella was dominated by C2 (30–37 mol%), GTX5 (12–19 mol%) and GTX6 (27–33 mol%) and was constant over the cell cycle. However, the net cellular PST production rate varied over the cell cycle and was highest (127 fmol cell−1 h−1) during the G2+M phase of the cell cycle (56% cells in G2+M), slowing considerably (−14 fmol cell−1 h−1) as cells entered the G1 phase (66% cells in G1). Quantitative real time PCR revealed that Sam, Sahh and Map were up-regulated (doubling in each case) during PST production and the G2+M phase of the cell division cycle before irradiance. Alexandrium catenella actively produced PSTs during this dark period, suggesting that photon irradiance was not a direct trigger for PST production. Interactions between cell division and PST production appear complex and the present study has demonstrated correlations between PST production and expression of Sam, Sahh and Map, which warrants further investigation.

Toxic Alexandrium minutum (Dinophyceae) from Vietnam with new gonyautoxin analogue

Clonal cultures of Alexandrium species collected from a shrimp pond on the northern coast of Vietnam were established and morphologically identified as Alexandrium minutum. Nucleotide sequences of domains 1 and 2 of the large subunit ribosomal (LSU) rRNA gene showed high sequence similarity to A. minutum isolates from Malaysia. Paralytic shellfish toxin profile of the clones was characterized by the dominance of GTX4, GTX1, and NEO. GTX3, GTX2, and dcSTX were also present in trace amount. Toxin content varied among the strains and growth stages, ranged from 3.0 to 12.5 fmol cell −1. In addition to these known toxin components, a new gonyautoxin derivative was detected by HPLC, eluting between GTX4 and GTX1. The peak of this compound disappeared under non-oxidizing HPLC condition but unchanged either after treated with 0.05 M ammonium phosphate/10% mercaptoethanol or 0.1N HCl hydrolysis. LCMS ion scanning showed a parental ion of [ M + H] + at m/ z 396, [ M − SO 3] + at m/ z 316, and [ M − SO 4] + at m/ z 298. Based on these results, the derivative was identified as deoxy-GTX4-12ol, and this represents the first report of this toxin analogue.